The evolutionary origins of peroxynitrite signalling.

Peroxynitrite is a reactive intermediate formed in vivo through uncatalysed reaction of superoxide and nitric oxide radicals. Despite significant interest in detecting peroxynitrite in vivo and understanding its production, little attention has been given to the evolutionary origins of peroxynitrite signalling. Herein we focus on two enzymes that are key to the biosynthesis of superoxide and nitric oxide, NADPH oxidase 5 (NOX5) and endothelial nitric oxide synthase (eNOS), respectively. Multiple sequence alignments of both enzymes including homologues from all domains of life, coupled with a phylogenetic analysis of NOX5, suggest eNOS and NOX5 are present in animals as the result of horizontal gene transfer from ancestral cyanobacteria to ancestral eukaryotes. Therefore, biochemical studies from other laboratories on a NOX5 homologue in Cylindrospermum stagnale and an eNOS homologue in Synechococcus sp. PCC 7335 are likely to be of relevance to human NOX5 and eNOS and to the production of superoxide, nitric oxide and peroxynitrite in humans.

Phytocannabinoid-dependent mTORC1 regulation is dependent upon inositol polyphosphate multikinase activity.

BACKGROUND AND PURPOSE: Cannabidiol (CBD) has been shown to differentially regulate the mechanistic target of rapamycin complex 1 (mTORC1) in preclinical models of disease, where it reduces activity in models of epilepsies and cancer and increases it in models of multiple sclerosis (MS) and psychosis. Here, we investigate the effects of phytocannabinoids on mTORC1 and define a molecular mechanism. EXPERIMENTAL APPROACH: A novel mechanism for phytocannabinoids was identified using the tractable model system, Dictyostelium discoideum. Using mouse embryonic fibroblasts, we further validate this new mechanism of action. We demonstrate clinical relevance using cells derived from healthy individuals and from people with MS (pwMS). KEY RESULTS: Both CBD and the more abundant cannabigerol (CBG) enhance mTORC1 activity in D. discoideum. We identify a mechanism for this effect involving inositol polyphosphate multikinase (IPMK), where elevated IPMK expression reverses the response to phytocannabinoids, decreasing mTORC1 activity upon treatment, providing new insight on phytocannabinoids' actions. We further validated this mechanism using mouse embryonic fibroblasts. Clinical relevance of this effect was shown in primary human peripheral blood mononuclear cells, where CBD and CBG treatment increased mTORC1 activity in cells derived from healthy individuals and decreased mTORC1 activity in cells derived from pwMS. CONCLUSION AND IMPLICATIONS: Our findings suggest that both CBD and the abundant CBG differentially regulate mTORC1 signalling through a mechanism dependent on the activity of the upstream IPMK signalling pathway, with potential relevance to the treatment of mTOR-related disorders, including MS.

A Dictyostelium discoideum mitochondrial fluorescent tagging vector that does not affect respiratory function.

Visualizing mitochondria in living Dictyostelium discoideum cells using fluorescent dyes is often problematic due to variability in staining, metabolism of the dyes, and unknown potential effects of the dyes on mitochondrial function. We show that fluorescent labelling of mitochondria, using an N-terminal mitochondrial localization sequence derived from the D. discoideum protein GcvH1 (glycine cleavage system H1) attached to a red fluorescent protein enables clear mitochondrial imaging. We also show that this labelling has no effect upon mitochondria load or respiratory function.

A new mechanism for cannabidiol in regulating the one-carbon cycle and methionine levels in Dictyostelium and in mammalian epilepsy models.

BACKGROUND AND PURPOSE: Epidiolex™, a form of highly purified cannabidiol (CBD) derived from Cannabis plants, has demonstrated seizure control activity in patients with Dravet syndrome, without a fully elucidated mechanism of action. We have employed an unbiased approach to investigate this mechanism at a cellular level. EXPERIMENTAL APPROACH: We use a tractable biomedical model organism, Dictyostelium, to identify a protein controlling the effect of CBD and characterize this mechanism. We then translate these results to a Dravet syndrome mouse model and an acute in vitro seizure model. KEY RESULTS: CBD activity is partially dependent upon the mitochondrial glycine cleavage system component, GcvH1 in Dictyostelium, orthologous to the human glycine cleavage system component H protein, which is functionally linked to folate one-carbon metabolism (FOCM). Analysis of FOCM components identified a mechanism for CBD in directly inhibiting methionine synthesis. Analysis of brain tissue from a Dravet syndrome mouse model also showed drastically altered levels of one-carbon components including methionine, and an in vitro rat seizure model showed an elevated level of methionine that is attenuated following CBD treatment. CONCLUSIONS AND IMPLICATIONS: Our results suggest a novel mechanism for CBD in the regulating methionine levels and identify altered one-carbon metabolism in Dravet syndrome and seizure activity.

A novel mechanism for the anticancer activity of aspirin and salicylates.

Epidemiological studies indicate that long­term aspirin usage reduces the incidence of colorectal cancer (CRC) and may protect against other non­CRC associated adenocarcinomas, including oesophageal cancer. A number of hypotheses have been proposed with respect to the molecular action of aspirin and other non­steroidal anti­inflammatory drugs in cancer development. The mechanism by which aspirin exhibits toxicity to CRC has been previously investigated by synthesising novel analogues and derivatives of aspirin in an effort to identify functionally significant moieties. Herein, an early effect of aspirin and aspirin­like analogues against the SW480 CRC cell line was investigated, with a particular focus on critical molecules in the epidermal growth factor (EGF) pathway. The present authors proposed that aspirin, diaspirin and analogues, and diflunisal (a salicylic acid derivative) may rapidly perturb EGF and EGF receptor (EGFR) internalisation. Upon longer incubations, the diaspirins and thioaspirins may inhibit EGFR phosphorylation at Tyr1045 and Tyr1173. It was additionally demonstrated, using a qualitative approach, that EGF internalisation in the SW480 cell line may be directed to endosomes by fumaryldiaspirin using early endosome antigen 1 as an early endosomal marker and that EGF internalisation may also be perturbed in oesophageal cell lines, suggestive of an effect not only restricted to CRC cells. Taken together and in light of our previous findings that the aspirin­like analogues can affect cyclin D1 expression and nuclear factor­κB localisation, it was hypothesized that aspirin and aspirin analogues significantly and swiftly perturb the EGFR axis and that the protective activity of aspirin may in part be explained by perturbed EGFR internalisation and activation. These findings may also have implications in understanding the inhibitory effect of aspirin and salicylates on wound healing, given the critical role of EGF in the response to tissue trauma.

The Cytotoxicity and Synergistic Potential of Aspirin and Aspirin Analogues Towards Oesophageal and Colorectal Cancer.

BACKGROUND: Oesophageal cancer (OC) is a deadly cancer because of its aggressive nature with survival rates that have barely improved in decades. Epidemiologic studies have shown that low-dose daily intake of aspirin can decrease the incidence of OC. METHODS: The toxicity of aspirin and aspirin derivatives to OC and a CRC cell line were investigated in the presence and absence of platins. RESULTS: The data in this study show the effects of a number of aspirin analogues and aspirin on OC cell lines that originally presented as squamous cell carcinoma (SSC) and adenocarcinoma (ADC). The aspirin analogues fumaryldiaspirin (PN517) and the benzoylsalicylates (PN524, PN528 and PN529), were observed to be more toxic against the OC cell lines than aspirin. Both quantitative and qualitative apoptosis experiments reveal that these compounds largely induce apoptosis, although some necrosis was evident with PN528 and PN529. Failure to recover following the treatment with these analogues emphasized that these drugs are largely cytotoxic in nature. The OE21 (SSC) and OE33 (ADC) cell lines were more sensitive to the aspirin analogues compared to the Flo-1 cell line (ADC). A non-cancerous oesophageal primary cells NOK2101, was used to determine the specificity of the aspirin analogues and cytotoxicity assays revealed that analogues PN528 and PN529 were selectively toxic to cancer cell lines, whereas PN508, PN517 and PN524 also induced cell death in NOK2101. In combination index testing synergistic interactions of the most promising compounds, including aspirin, with cisplatin, oxaliplatin and carboplatin against the OE33 cell line and the SW480 colorectal cancer (CRC) cell line were investigated. Compounds PN517 and PN524, and to a lesser extent PN528, synergised with cisplatin against OE33 cells. Cisplatin and oxaliplatin synergised with aspirin and PN517 when tested against the SW480 cell line. CONCLUSION: These findings indicate the potential and limitations of aspirin and aspirin analogues as chemotherapeutic agents against OC and CRC when combined with platins.

Changes in defensive functioning in completed psychoanalyses: the penn psychoanalytic treatment collection.

Theory predicts that patients completing psychoanalysis should improve in their dynamic functioning. The aim of this naturalistic study is to examine whether a sample of 17 subjects from the Penn Psychoanalytic Treatment Collection with completed, tape-recorded psychoanalyses demonstrated improvement in one dynamic aspect: their defense mechanisms. The pre-post effect size for the change in overall defensive functioning (ODF) of the sample was large (.76) and statistically significant (p = .01). The percentage of subjects who improved in their ODF (71%) was similar to that found by others who studied the same sample using general functioning measures. These findings provide the first empirical evidence to support a trait-like change in dynamic personality functioning in patients who have undergone psychoanalysis. Randomized controlled studies with homogeneous samples are needed to further confirm these findings.

Improvement and recovery from suicidal and self-destructive phenomena in treatment-refractory disorders.

Long-term data about recovery from suicidal and self-destructive phenomena are limited. We examined these phenomena in a naturalistic, follow-along study of 226 adults with treatment-refractory disorders admitted at the Austen Riggs Center. Follow-along interviews systematically rated suicidal ideation, suicide attempts, self-mutilation, and other self-destructive phenomena. We identified vital status in 98.7%, finding 1 death by suicide. Among 79 who made at least 1 suicide attempt in the 6 months before admission, 61 (77.2%) were estimated to be free of suicidal acts by a median of 7.18 years. Self-mutilation and other self-destructive phenomena showed slightly longer times to recovery. Among 156 individuals with suicidal ideation, 79 (50.6%) attained sustained recovery at a median of 8.69 years. On average 2.9 to 5.2 years were required from the last observed self-destructive event to attain sustained recovery. Most participants showed significant improvement in suicidal phenomena, whereas between 50.6% and 77.2% attained sustained recoveries.